Methods for analysis of free and autoantibody-bound biomarkers and associated compositions, devices, and systems

ABSTRACT

The present invention provides methods, compositions, and kits associated with analyzing, enriching, and/or isolating a biomarker or analyte in a biological sample. In one aspect, for example, a method for determining a concentration of a biomarker in a biological sample can include binding any unbound biomarker with an antibody specific for the biomarker to form antibody-bound biomarker, enriching the antibody-bound biomarker and any endogenous autoantibody-bound biomarker to form an enriched fraction, identifying the biomarker in the enriched fraction, and determining the concentration of the biomarker in the biological sample. In one aspect, the concentration of the biomarker is derived from initially unbound biomarker and autoantibody-bound biomarker in the biological sample.

PRIORITY DATA

This application is a continuation of U.S. patent application Ser. No.13/676,980, filed Nov. 14, 2012, now issued as U.S. Pat. No. 9,140,695,which claims the benefit of U.S. Provisional Patent Application Ser. No.61/632,125, filed on Jan. 18, 2012, each of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

The presence or absence of specific biomarkers can be predictive in thediagnosis and/or determination of a variety of conditions in anindividual. As such, the accuracy of detection and quantification of aparticular biomarker can be important for the correct diagnosis andtreatment of an associated condition. In those cases where a biomarkeris incorrectly identified in or its concentration is incorrectlydetermined in a biological sample, the individual can be improperlytreated for a condition that is not present or can be left untreated fora condition that is present.

A variety of situations can make the detection of a biomarkerproblematic, both for the presence or absence of the biomarker, as wellas the concentration level of the biomarker in the individual. One suchsituation can occur for individuals expressing autoantibodies to abiomarker in question. In such cases, the autoantibodies (AAb) canadversely affect the assay used to detect the biomarker. As one example,the measurement of thyroglobulin (Tg) is commonly used for the follow-upof patients treated for differentiated thyroid carcinoma (TC). Becausethyroid tissue is the only source of Tg, total thyroidectomy andradioactive ablation should decrease serum concentrations of Tg to verylow or undetectable levels. A rise of serum concentration of Tg inpost-treatment patients is thus indicative of the recurrence of the TC.In a retrospective study on assessment of the utility of multiplepotential biomarkers of the recurrence of TC, post-treatment Tgconcentration was found to be the strongest independent predictor of therecurrence. One difficulty in using Tg as a biomarker is related to thepresence of anti-Tg autoantibodies (Tg-AAb) in the blood of certainindividuals. Tg antibodies were first described by Stokinger andHeidelberger in 1937, and subsequent research to date has not generatedan effective commercial technique to overcome interference of Tg-AAb intesting for Tg. Current protocols to assess reliability of Tgmeasurements with immunoassays (IA) commonly test every specimenanalyzed for Tg also for the presence of Tg-AAb.

Furthermore, it has been hypothesized that there might be a pathologicaland causative link between thyroid cancer coexistence and thyroidautoimmunity. Approximately 25% of patients with TC and up to 10% ofindividuals without TC are positive for Tg-AAb, and thus the presence ofautoantibodies in samples tested for Tg by IA could cause false-negativeresults when epitopes used by the capture or detection antibody areoccupied by the Tg-AAb. This can potentially cause misdiagnosis of therecurrence of TC.

SUMMARY

The present disclosure provides methods for analyzing, enriching, and/orisolating a biomarker or analyte in a biological sample, whether thesample contains autoantibody-bound biomarker, autoantibody-freebiomarker (not bound to autoantibody), or both autoantibody-boundbiomarker and autoantibody-free biomarker. The present scopeadditionally includes compositions, devices, kits, and systems relatingto such analysis, enrichment, and/or isolation. In one aspect, forexample, a method for determining a concentration of a biomarker in abiological sample is provided. Such a method can include binding anyunbound biomarker with an antibody specific for the biomarker to formantibody-bound biomarker complexes, enriching the antibody-boundbiomarker and any endogenous autoantibody-bound biomarker to form anenriched fraction, identifying the biomarker in the enriched fraction,and determining the concentration of the biomarker in the biologicalsample. In one aspect, the concentration of the biomarker is derivedfrom initially unbound biomarker and autoantibody-bound biomarker in thebiological sample.

Various techniques for enriching the biomarker are contemplated, and anysuch technique is considered to be within the present scope. In oneaspect, identifying the biomarker in the enriched fraction can furtherinclude digesting the antibody-bound biomarker and the endogenousautoantibody-bound biomarker complexes and detecting a product ofdigestion to identify and quantify the biomarker. In one specificaspect, determining the concentration of the biomarker in the biologicalsample can include detecting the concentration of the product ofdigestion in the enriched fraction. In another aspect, enriching canfurther include enriching the antibody-bound biomarker and anyendogenous autoantibody-bound biomarker to form an enriched fraction. Inyet another aspect, enriching can include precipitating theantibody-bound biomarker and any endogenous autoantibody-boundbiomarker. In yet another aspect, enriching can include separation ofthe biomarker from the biological sample using anti-biomarker antibody,protein A, protein G, protein L, anti-immunoglobulin antibody, or acombination thereof. In a further aspect, enriching can includeprecipitating the antibody-bound biomarker and any endogenousautoantibody-bound biomarker along with a chemical precipitation ofimmunoglobulins, or alternatively unrelated proteins can be separatedfrom solution by a chemical precipitation, while the antibody-bound andautoantibody-bound biomarker is kept in solution.

Various biomarkers are contemplated, and any such biomarker for whichautoantibodies can negatively affect a detection assay is considered tobe within the present scope. Non-limiting examples of such biomarkerscan include thyroglobulin, insulin, cardiac troponin I, tumor suppressorprotein p53, tumor-associated antigens, anti-retinal autoantibodies,markers of age-related macular degeneration, and the like. In onespecific aspect, the biomarker is thyroglobulin.

In another aspect, identifying the biomarker can be an immune basedmethod such as, for example, RIA, ELISA, EMIT, chemiluminescenceimmunoassay, or the like, including combinations thereof. In yet anotheraspect, identifying the biomarker can be performed using massspectrometry. One specific example of mass spectrometry that may beuseful is tandem mass spectrometry; another example is high massaccuracy/high mass resolution mass spectrometry (e.g. Orbitrap™, ThermoScientific).

The present disclosure additionally provides a method for determining aconcentration of thyroglobulin in a biological sample. Such a method caninclude binding any unbound thyroglobulin with an antibody specific forthyroglobulin to form antibody-bound thyroglobulin, enriching theantibody-bound thyroglobulin and any endogenous autoantibody-boundthyroglobulin to form an enriched fraction, identifying thethyroglobulin in the enriched fraction, and determining theconcentration of the thyroglobulin in the biological sample.

In one aspect, identifying the thyroglobulin in the enriched fractioncan further include digesting the antibody-bound thyroglobulin and theendogenous autoantibody-bound thyroglobulin complexes, and detecting aproduct of digestion to identify thyroglobulin. Non-limiting examples ofproducts of thyroglobulin digestion can include SEQ ID 001, SEQ ID 002,SEQ ID 003, SEQ ID 004, SEQ ID 005, or a combination thereof.

The present disclosure additionally provides a kit for determining aconcentration of thyroglobulin in a biological sample. Such a kit caninclude a housing, an antibody preparation including at least one firstantibody specific for thyroglobulin, magnetic beads having at least onesecond antibody coupled thereto, the antibody specific to at least oneepitope of a thyroglobulin fragment, and instructions describing usingthe antibody preparation and the magnetic beads, the instructions alsodescribing enrichment and digestion of thyroglobulin. In one specificaspect, the second antibody is specific for SEQ ID 001.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a provides results showing the total protein content betweenprecipitates and supernatants according to one aspect of the presentdisclosure.

FIG. 1 b provides results showing the total protein content anddistribution of IG between precipitates and supernatants according toone aspect of the present disclosure.

FIG. 1 c provides results showing the total protein content anddistribution of albumin between precipitates and supernatants accordingto one aspect of the present disclosure.

FIG. 2 a provides results showing the percent distribution ofthyroglobulin between the supernatants and the precipitates inbiological sample for no antibody (NA), Tg-autoantibody (AAb), andpolyclonal rabbit antibody (PRAb) added to the sample with ratio ofammonium sulfate/serum 0.65:1 according to one aspect of the presentdisclosure.

FIG. 2 b provides results showing the percent distribution ofthyroglobulin between the supernatants and the precipitates inbiological sample for no antibody (NA), Tg-autoantibody (AAb), andpolyclonal rabbit antibody (PRAb) added to the sample with ratio ofammonium sulfate/serum 1:1 according to one aspect of the presentdisclosure.

FIG. 2 c provides results showing the percent distribution ofthyroglobulin between the supernatants and the precipitates inbiological samples for no antibody (NA), Tg-autoantibody (AAb), andpolyclonal rabbit antibody (PRAb) added to the sample with ratio ofammonium sulfate/serum 2:1 according to one aspect of the presentdisclosure.

FIG. 3 provides a product ion mass spectrum of thyroglobulin-specificpeptide VIFDANAPVAVR (SEQ ID 001) according to one aspect of the presentdisclosure.

FIG. 4 shows chromatograms of biological samples containing 5 ng/mL ofthyroglobulin according to one aspect of the present disclosure.

FIG. 5 shows results of LC-MS/MS method comparison with a BeckmanCoulter Access analyzer for thyroglobulin autoantibody negative (A, B)and thyroglobulin autoantibody positive samples (C). Thyroglobulinconcentrations 0-350 ng/mL (A), thyroglobulin concentrations below 60ng/mL (B); according to one aspect of the present disclosure.

DETAILED DESCRIPTION

Before the present invention is disclosed and described, it is to beunderstood that this invention is not limited to the particularstructures, process steps, or materials disclosed herein, but isextended to equivalents thereof as would be recognized by thoseordinarily skilled in the relevant arts. It should also be understoodthat terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an antibody” includes one or more of such antibodies orsynthetic binders, and reference to “the protein” includes reference toone or more of such proteins or small molecular biomarkers.

DEFINITIONS

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

As used herein, the term “autoantibody” refers to an antibody producedby the immune system of a subject that is directed against one or moreof the subject's own biomarkers.

As used herein, the term “synthetic binder” refers to an aptamer,synthetic binding site, cofactor, or combinations of the above.

As used herein, the term “free” and “unbound” when used in reference toa biomarker's association with an antibody can be used interchangeably,and relate to a biomarker in a biological sample or in a subject that isnot complexed or otherwise bound to an autoantibody specific for thebiomarker. Thus, in some aspects a “free biomarker” or an “unboundbiomarker” can refer to a biomarker that is not bound by any antibodywhatsoever. In other aspects, these terms can refer to a biomarker thatis not bound by autoantibody, but may be bound by antibody introducedinto a biological sample according to the present methods.

As used herein, the term “antibody” refers to an immunoglobulinspecifically immunoreactive to a given antigen. The term “antibody” isintended to include whole antibodies of any isotype (IgG, IgA, IgM, IgE,IgY, etc.), and fragments thereof. It is noted that antibodies can bederived from a variety of animal species, and that antibodies from anyspecies that can be beneficially utilized according to aspects of thepresent disclosure are considered to be within the present scope. Insome non-limiting aspects, shark and/or camelid antibodies can be used.An “antibody” of the present invention also includes an antibodypreparation. In addition to this traditional definition, in some aspectsthe term “antibody” can be used to encompass other molecules andstructures that can serve a similar binding purpose. Non-limitingexamples of non-traditional antibodies can include proteins, aptamers,synthetic binding sites, cofactors, and the like, including combinationsthereof.

As used herein, the term “subject” refers to a mammal that may benefitfrom the techniques according to aspects of the present disclosure.Examples of subjects include humans, and may also include other animalssuch as horses, pigs, cattle, dogs, cats, rabbits, and aquatic mammals.

As used herein, the term “biological sample” refers to any sample of abiological nature obtained from a subject that may contain a biomarker.Non-limiting examples include biological fluids and biological tissuessuch as blood, blood serum, plasma, saliva, semen, vaginal fluid, lymph,urine, lachrymal fluid, cancerous tissue, non-cancerous tissue, tumortissue, skin tissue, and the like.

As used herein, the term “substantially” refers to the complete ornearly complete extent or degree of an action, characteristic, property,state, structure, item, or result. For example, an object that is“substantially” enclosed would mean that the object is either completelyenclosed or nearly completely enclosed. The exact allowable degree ofdeviation from absolute completeness may in some cases depend on thespecific context. However, generally speaking the nearness of completionwill be so as to have the same overall result as if absolute and totalcompletion were obtained. The use of “substantially” is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a composition that is“substantially free of” particles would either completely lackparticles, or so nearly completely lack particles that the effect wouldbe the same as if it completely lacked particles. In other words, acomposition that is “substantially free of” an ingredient or element maystill actually contain such item as long as there is no measurableeffect thereof.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 1 to about 5” should beinterpreted to include not only the explicitly recited values of about 1to about 5, but also include individual values and sub-ranges within theindicated range. Thus, included in this numerical range are individualvalues such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4,and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. Thissame principle applies to ranges reciting only one numerical value as aminimum or a maximum. Furthermore, such an interpretation should applyregardless of the breadth of the range or the characteristics beingdescribed.

Invention

The present disclosure provides methods for analyzing, enriching, and/orisolating a biomarker or analyte in a biological sample, whether thesample contains autoantibody-bound biomarker, autoantibody-freebiomarker, or both autoantibody-bound biomarker and autoantibody-freebiomarker. The present scope additionally includes compositions,devices, kits, and systems relating to such analysis, enrichment, and/orisolation.

In many traditional peptide purification schemes, the target peptide isusually used as bait to capture an antibody directed to a targetanalyte. The target peptide is generally separated from the antibody insubsequent processing; however in some cases the target peptide may notbe entirely removed. This can interfere with subsequent analysis becausethe target peptide is an impurity that can be chemically the same as thetarget analyte. The purification schemes according to aspects of thepresent disclosure overcome this problem by using a modified bait. Forexample, if the bait is an isotopically labeled version of the targetanalyte, then it can be separated from the target analyte by massspectrometry. Possible interference from the internal standard can alsobe avoided by using a labeled bait having a different mass from theinternal standard. Similarly, if the bait is an extended sequence of thetarget analyte, then the bait should not interfere with a subsequentanalysis by mass spec.

As has been described, the accuracy of an assay for the detection of abiomarker in a subject can be greatly reduced by the presence ofautoantibodies directed to the biomarker. In some cases an assay canshow and absence of the biomarker when it is in fact present in thebiological sample, thus generating a false-negative result. Theinventors have discovered that the addition of antibody specific for thebiomarker to the biological sample prior to enrichment can actuallyincrease the accuracy of biomarker detection is such assays. Thiscounter-intuitive addition of antibody can increase biomarker enrichmentto a degree that allows clinical-level diagnostic results with increasedaccuracy and degree of confidence.

In one aspect, for example, a method for determining a total amount of abiomarker in a biological sample is provided. It is intended that thescope of this method include the testing of biological samples having nobiomarker, free biomarker (i.e. not bound by autoantibody), and/orautoantibody-bound biomarker. In other words, the present methods allowthe clinically accurate assaying of a biological sample for thosesubjects having autoantibodies for the biomarker and for those subjectslacking autoantibodies for the biomarker. Thus, a negative resultdemonstrates the actual absence of the biomarker in the subject asopposed to a potential false-negative result whereby the biomarker ispresent but bound to autoantibodies.

Various method steps are contemplated to accomplish this enhancedbiomarker detection assay. In one aspect, for example, the method caninclude binding any unbound biomarker with an antibody specific for thebiomarker to form antibody-bound biomarker, and enriching theantibody-bound biomarker and any endogenous autoantibody-bound biomarkerto form an enriched fraction. In some aspects, the total amount of thebiomarker in the enriched fraction can include antibody-bound biomarkerwhere the antibody includes only antibodies added to the biologicalsample in vitro. In other aspects, the total amount of the biomarker inthe enriched fraction can include AAb-bound biomarker and antibody-boundbiomarker (i.e. the added antibody). In yet other aspects, the totalamount of the biomarker in the enriched fraction can include onlyAAb-bound biomarker. Additionally, in some aspects the biological samplecan include no biomarker or an amount of biomarker that is below thedetection threshold. It is thus contemplated that the present methodincludes the testing of biological samples that test negative for thepresence of the biomarker.

Once the enriched fraction has been obtained, the biomarker can bedetected in the fraction. In one aspect, detection can thus includeverification of the presence of the biomarker in the enriched fraction.In another aspect, detection can also include a determination of theamount or concentration of biomarker in the enriched fraction. As such,the total concentration of biomarker can be determined from theproportion obtained in the enriched fraction.

Various biomarkers are contemplated for detection and/or analysisaccording to aspects of the present disclosure. It is noted that anybiomarker for which autoantibodies may affect detection and/ormeasurement is considered to be within the present scope. For example,the biomarker can include, without limitation, biopolymers, proteins,peptides, small molecules, and the like. Specific non-limiting examplesof such biomarkers can include thyroglobulin, insulin, cardiac troponinI, tumor suppressor protein p53, tumor-associated antigens, anti-retinalautoantibodies, markers of age-related macular degeneration, and thelike.

Thyroglobulin (Tg) is one example of a biomarker that can bebeneficially assayed according to aspects of the present disclosure. Itshould be noted that the following description and discussion relatingto Tg is exemplary, and that any biomarker that can be so assayed isconsidered to be within the present scope. As has been described,anti-Tg autoantibodies (Tg-AAb) in the blood of certain individuals canaffect the accuracy of Tg biomarker testing using traditional assaymethods. The current techniques overcome the interference of Tg-AAb andallow the quantification of Tg in a biological sample that is specific,sensitive, and robust enough for routine use in clinical laboratories.

As has been described, one difficulty for traditional assay techniquesin the analysis of Tg in a biological sample is related to multipleforms of Tg that can be present in circulating blood, namely the freeand Tg-AAb bound Tg. It has been shown that at least nineteen epitopeson the Tg molecule could participate in autoantibody binding.Considering the inter-individual differences in these epitopes, alongwith the affinity of Tg-AAbs towards Tg, and the task of measuring Tg(and other biomarkers for which AAb can be present in circulation) canbe very complex, in some cases more complex than for many otherendogenous biomarkers. Because the presence of Tg (free or AAb-bound) isindicative of the recurrence of thyroid cancer (TC), low concentrationsof total Tg (free and AAb-bound) need to be accurately measured, and theexistence in samples of multiple forms of Tg and Tg-AAb complicates thetask for traditional methods. In many cases, concentrations of Tg above1 ng/mL can be considered as indicative of the recurrence of thyroidcancer in such patients. In one aspect, Tg can be measured inconcentrations at least as low as 0.5 ng/mL using techniques accordingto aspects of the present disclosure.

As has been described, free biomarker and any AAb-bound biomarkerpresent in the biological sample can be enriched by binding any free orunbound biomarker with an antibody capable of forming a complex with thebiomarker. Any form or type of antibody specific for the biomarker canbe utilized. In one aspect, for example, the antibody can be polyclonal,monoclonal, or a mixture thereof. Additionally, the antibody can bederived from any appropriate species, including, without limitation,rabbit, goat, chicken, mouse, fish, cartilaginous fish, camelid, human,and the like, including combinations thereof.

Biomarker enrichment can be performed by any technique that facilitatesthe pooling of biomarker that is not AAb-bound and any AAb-boundbiomarker present into a total biomarker concentration or amount. Byconcentrating the Abb-bound biomarker and the free biomarker into asingle fraction, total concentration of the biomarker in a sample can bedetermined. Additionally, in some cases all biomarkers in the biologicalsample can be bound by biomarker-AAb, and little to no free biomarker ispresent in circulation.

Various techniques for enrichment of a biomarker can be utilized, andany such technique that facilitates the pooling of free biomarker withAAb-bound biomarker is considered to be within the present scope. In oneaspect, for example, pooling of the free and AAb-bound complexes can beaccomplished by binding the free biomarker with antibody and enrichingby maintaining the pooled complexes in solution while unrelatedconstituents from the biological sample are separated from solution. Asone specific example, unrelated proteins can be separated from sample bya chemical precipitation, while maintaining the biomarker-antibody andbiomarker-AAb complexes in solution. One non-limiting example of such achemical precipitation can include caprylic acid, however any reagentcapable of precipitating albumin and other abundant proteins whileallowing Tg and IG to remain in solution is considered to be within thepresent scope.

In another aspect, the pooled complexes can be enriched and separatedfrom solution by precipitation. Various techniques are contemplated tofacilitate precipitation or separation of the pooled complexes.Non-limiting examples can include precipitation techniques utilizinganti-biomarker antibody, protein A, protein G, protein L,anti-immunoglobulin antibodies, or a combination thereof. In onespecific aspect, enrichment can be accomplished by coprecipitating Tgwith a precipitation of IG, such as, for example, an ammonium sulfateprecipitation. As such, Tg-complexes can be produced as has beendescribed using any antibody capable of forming a complex with Tg. SuchTg-antibody complexes can be coprecipitated along with IG.

Returning to Tg, enrichment can be based on the discovered phenomenonthat substantially all forms of Tg present in a biological sample can beconcentrated in a single fraction, while at the same time a largefraction of non-targeted blood proteins can be depleted from thesamples. Various strategies that can be used to enrich Tg are describedabove. As one specific example, enrichment from a biological sample canbe accomplished using Affinity-Assisted Precipitation (AAP). As has beendescribed, anti-Tg antibodies are added to the biological sample toAb-bind any unbound Tg biomarker. Following binding of any unbound orfree Tg with Tg-Ab, both AAb-bound Tg and Ab-bound Tg (i.e. the pooledcomplexes) can be co-precipitated with ammonium sulfate along with IG.As such, the addition of anti-Tg antibody of any species to the samplecan enhance Tg precipitation, thus increasing the accuracy of thedetection assay. The supernatant following precipitation can bediscarded and the Tg-containing precipitates can be further processed.Effects of concentration of the antibody and amount of ammonium sulfateon distribution of Tg, total protein, albumin, and IgG between thesupernatants and the precipitates are shown in FIGS. 1 and 2.

FIG. 1 a-c show the total protein content and distribution of Tg and IGbetween precipitates and supernatants evaluated in samples containingand not containing Tg-AAb using different ammonium sulfate/serum ratios,with and without anti-Tg antibodies. Percent distribution of totalproteins (FIG. 1 a), IgG (FIG. 1 b), and albumin (FIG. 1 c) between thesupernatants and the precipitates are shown.

FIG. 2 a-c show thyroglobulin concentration measured by LC-MS/MS method.The percent distribution of thyroglobulin between the supernatants andthe precipitates in the biological samples are shown for no antibody(NA), Tg-autoantibody (AAb), and polyclonal rabbit antibody (PRAb) addedto the sample. In FIG. 2 a the ratio of ammonium sulfate to serum is0.65:1; in FIG. 2 b the ratio of ammonium sulfate to serum is 1:1; andin FIG. 2 c the ratio of ammonium sulfate to serum is 2:1.

It is thus observed that in serum and plasma samples containing Tg-AAb,Tg bound to AAb is separated into the IG-containing fraction. Higherratios of ammonium sulfate to serum/plasma increase efficiency of Tgprecipitation, also resulting in enhanced precipitation of otherproteins. A large fraction of Tg in samples of patients not havingTg-AAb also precipitated (FIG. 1); this may be through co-precipitationwith IG, such as precipitation of a Tg/antibody complex formed bynon-specific binding of antibody to Tg. In samples not containing IG, Tgdid not precipitate and remained in solution.

Adding anti-Tg antibodies to biological samples can thus increase theefficiency of the enrichment. Without intending to be bound to anyscientific theory, this may be due to the binding of free Tg to theexogenous antibody and partitioning of the antibody-Tg complexes intothe IG fraction. As an example, Tg enrichment based on precipitation ofTg with IG using ammonium sulfate can increase Tg enrichment to clinicallevels, while at the same time reducing the total concentration ofproteins by up to 70%. The ratio of ammonium sulfate to serum/plasmaduring Tg precipitation can be adjusted to assure completeness ofprecipitation of Tg, while minimizing precipitation of albumin and otherserum/plasma proteins as is shown in FIGS. 1 a-c and 2 a-c.Additionally, it is noted that enrichment via the addition of antibodywas particularly enhanced in samples containing higher concentrations ofTg, such as, for example, above 50 ng/ml.

The ammonium sulfate precipitation produces a powdery precipitate that,after centrifugation, results in a dense protein pellet. Once thesupernatant is removed, the precipitate is easily re-dissolved in water.As such, this precipitation enrichment concentrates all forms of Tgpresent in serum (free and AAb-bound) into a single fraction, reducessample complexity (albumin and large fraction of serum/plasma proteinsare removed with the supernatant), and allows sample matrix exchangeprior to subsequent sample processing.

Following enrichment, the sample can be processed to varying degrees. Inone aspect, for example, the biomarker can be directly assayed from theenriched fraction. In another aspect, the enriched fraction can befurther processed to facilitate biomarker detection and/orquantification. In one specific example relating to Tg, re-dissolvedenriched biomarker can be denatured, reduced, and digested with, forexample, sodium deoxycholate (DOC), dithiothreitol (DTT), Trypsin,endoproteinase Lys-C, endoproteinase Asp-N, thrombin, and the like,including combinations thereof.

In some cases, the concentration of DOC can affect the extent ofdenaturing and the recovery of the targeted peptide. Lack of DOC canlead to incomplete digestion, while excess DOC can increase sampleviscosity, thus affecting the immunoaffinity enrichment of thebiomarker. The denaturing, reduction, and digestion can be optimized tofacilitate rapid, complete, and reproducible digestion of Tg.

Following digestion, the sample can be processed to varying degrees. Inone aspect, for example, the biomarker can be directly assayed from thedigested fraction. In another aspect, the enriched fraction can befurther processed to facilitate biomarker detection and/orquantification. For example, protein can be further separated to improvethe processing and detection of the biomarker or digested portions ofthe biomarker. In one aspect, for example, chromatographic separationcan be utilized to further enrich the biomarker or biomarker fragments.Various chromatography techniques are known, and any such technique isconsidered to be within the present scope. In one specific aspect,one-dimensional reversed phase chromatography can be used. While suchtechniques can be useful depending on the biomarker and specifics of thegiven separation procedure, in other aspects multidimensionalseparations can be used to improve signal-to-noise ratio and reducebackground noise. In one aspect, two-dimensional separation can beoptimized for the targeted biomarker using two chromatographic columnsusing complementary retention mechanisms with second dimensionseparation under ultra-high performance liquid chromatography (UPLC)conditions. In one specific aspect, good selectivity can be achieved bycombining a one-dimensional column with weak retention to atwo-dimensional column strongly retaining the peptide. The separationconditions can be further optimized to provide a good separation fromother endogenous peaks, while maintaining a short analysis time.Additionally, in some aspects other types of separation, in addition tothe one-dimensional separation described above, can be utilized as well.Non-limiting examples of such separation techniques includeelectrophoresis, capillary electrophoresis, nanoHPLC separation, slabgel electrophoresis, ion mobility, or any combination of the techniquesin either an online or offline format.

The complexity of the biological sample, the enriched fraction, thedigested fraction, or any sample following subsequent separation canalso be further reduced by a variety of techniques. Non-limitingexamples can include immunoaffinity depletion (for the removal ofabundant proteins) and immunoaffinity capture (to isolate or enrich thebiomarker or biomarker fragments), which can be used to reducecomplexity of biological samples. Other techniques can utilize onlineand offline immunoaffinity enrichment with antibody immobilized onpolymeric and/or magnetic beads. Such techniques can be utilized toseparate the targeted products from the sample, or they can be used toseparate non-targeted products from the sample. Compared to onlineapproaches, off-line immunoaffinity enrichment can provide increasedflexibility in terms of selection of carriers, antibodies, assayformats, and experimental conditions.

In one specific aspect, Tg or a Tg product can be enriched from thetryptic digests using anti-peptide antibody conjugated to magneticbeads. Following elution from the beads, the products can be analyzed todetermine the presence and concentration in the sample. In one aspect,the analysis can be by a 2D-LC-MS/MS method.

As has been described, biomarker detection can be accomplished using allof the biomarker, substantially all of the biomarker, or a fragment ofthe biomarker. The present scope is not, therefore, limited to specificbiomarkers or portions of biomarkers. In the case of Tg, the sequencesdescribed herein are merely exemplary, and any fragment detectable withsome form of antibody is considered to be within the present scope. Asone example, immunoaffinity enrichment with polyclonal anti-peptideantibody showed binding for the capture of VIFDANAPVAVR peptide (SEQ ID001) from serum digests with high recovery. Non-limiting examples ofother peptide fragments from Tg can include FSPDDSAGASALLR (SEQ ID 002),LGDQEFIK (SEQ ID 003), FPLGESFLVAK (SEQ ID 004), and the like.

Enrichment conditions can be optimized by varying the amount ofantibody, magnetic beads, serum volume, composition and conditions ofthe capture, the washes and the elution. Additionally, interference fromnonspecifically bound peptides can be resolved by optimizing the washand the elution conditions. The immunoaffinity enrichment allowsselective capture and elution of the peptide resulting in substantialenhancement of the sensitivity and specificity of the method.

Accordingly, in one specific aspect an assay format can include (i)addition to biological samples of anti-Tg antibody; (ii) enrichment ofTg through an enrichment technique such as precipitation; (iii)re-dissolving the precipitate with a solvent containing internalstandard; (iv) denaturing, reducing, and digesting; (v) enrichment ofthe biomarker or biomarker fragments using anti-peptide antibodyconjugated to magnetic beads; and (vi) removal of non-specifically boundpeptides by washing the beads. In some aspects the method can alsoinclude (vii) elution of the targeted biomarker or biomarker fragment,followed by (viii) biomarker analysis, such as by 2DLC-MS/MS.

Regarding HPLC separation, it is noted that a variety of techniques canbe utilized and all such techniques are considered to be within thepresent scope. In one aspect, for example, a first separation can beperformed on an HPLC column that weakly retains a target analyte, and asecond separation can be performed on a column that strongly retains thetarget analyte.

It is noted that any technique for detecting a biomarker or biomarkerfragment capable of use with aspects of the present disclosure areconsidered to be within the present scope. In one aspect, for example,the detection can include an immune-based method such as, for example,RIA, ELISA, EMIT, chemiluminescence immunoassay, and the like, includingcombinations thereof. In another aspect, mass spectrometry can be used.Tandem mass spectrometry, for example, can be used for quantitativeanalysis of peptides in biological samples due to high sensitivity andspecificity. High mass accuracy/high mass resolution mass spectrometers(e.g. Orbitrap™, Thermo Scientific) also can be utilized for analysis.Generally, a product of digestion can be purified using separationtechniques and ionized to generate ions detectable by mass spectrometry,where the concentration of peptides is determined by mass spectrometry,and amount detected is related to the amount of biomarker in the testsample. The ions can be single charged or multiple charged. In oneaspect, ions selected in the first stage of mass analysis can bemonoisotopic or isotopic. In another aspect, ions selected in the secondstage of mass analysis can be monoisotopic or isotopic. Additionally, itis contemplated that in some cases ions selected in all following stagesof mass analysis can be monoisotopic or isotopic.

As an example, product ion mass spectrum of the Tg-specific peptideVIFDANAPVAVR (SEQ ID 001) is shown in FIG. 3. The solid arrow points toa double-charged parent ion, and the outlined arrows point to the majorproduct ions. Additionally, multiple reaction monitoring chromatogramsof the mass transitions of VIFDANAPVAVR (SEQ ID 001) are shown in FIG. 4in biological samples containing 5 ng/mL of thyroglobulin. Masstransitions of the VIFDANAPVAVR (SEQ ID 001) peptide are m/z636.36/1059.56 (A), 636.36/912.49 (B), 636.36/541.35 (C); and theinternal standard m/z 639.34/1065.56 (D), 639.84/1066.56 (E),639.34/918.48, 639.34/547.34 (F).

In one aspect, a detected ion can have an m/z ratio of 636.3595+/−0.5,636.8634+/−0.5, 637.3673+/−0.5, 637.8712+/−0.5, or a combinationthereof. In another aspect, one or more detected ions can bemonoisotopic or isotopic ions such as, for example, ions with m/z of541.3457+/−0.5, 542.3535+/−0.5, 543.3613+/−0.5, 544.3691+/−0.5;612.3828+/−0.5, 613.3906+/−0.5, 614.3984+/−0.5, 615.406275+/−0.5;726.4257+/−0.5, 727.4335+/−0.5, 728.4413+/−0.5, 729.4491+/−0.5;797.4628+/−0.5, 798.4706+/−0.5, 799.4784+/−0.5, 800.4862+/−0.5;912.4898+/−0.5, 913.4976+/−0.5, 914.5054+/−0.5, 915.5132+/−0.5;1059.5582+/−0.5, 1060.5660+/−0.5, 1061.5738+/−0.5, 1062.5816+/−0.5;1172.6422+/−0.5, 1173.6500+/−0.5, 1174.6578+/−0.5, 1175.6656+/−0.5, or acombination thereof.

Data of experiments of the evaluation of the assay imprecision aresummarized in Table 1. The total assay imprecision at the evaluatedconcentrations was less than 10%; imprecision of Tg measurements in QCsamples analyzed over 16 days was <14%. The LOQ and LOD of the methodwere 0.5 and 0.25 ng/mL (0.76 and 0.38 fmol/mL of the Tg dimer),respectively. The method was linear up to 1045 pg/mL with inaccuracy atthe highest level <10%. No carryover was observed following an injectionof a blank sample after a standard containing 1045 ng/mL (1.58 pmol/mL)of Tg.

TABLE 1 Within-run, between-run and total imprecision of LC-MS/MS methodfor thyroglobulin. Concentration, Within- Between- Sample ng/mL run, %run/day, % Total, % Low 1^(#) 2.1 6.75 3.67 7.69 Low QC 1* 2.3 na 13.9na Low 2^(#) 5.7 6.87 5.96 9.10 Medium QC 2* 6.5 na 10.5 na Medium^(#)14.8 6.56 5.40 8.50 High^(#) 399 3.56 1.71 3.95 High QC* 172.8 na 3.5 na^(#)Samples analyzed in three replicates per day over five days *Samplesanalyzed in one replicate per day, over 20 days

As such, a biomarker fragment can be used as a surrogate marker for thequantification of the biomarker itself. In the case of Tg, for example,VIFDANAPVAVR (SEQ ID 001) can used as a surrogate marker forquantification of Tg (FIG. 4). Advantages of the VIFDANAPVAVR (SEQ ID001) peptide for quantitation are in the ability to achieve aquantitative yield of the peptide with tryptic digestion, and in theabsence in its sequence of amino acids that could bepost-translationally modified.

In another aspect, thyroglobulin from the biological sample can bedigested to form peptide FSPDDSAGASALLR (SEQ ID 002). In this case, adetected ion can be monoisotopic or isotopic and have an m/z ratio of703.8497+/−0.5, 704.3536+/−0.5, 704.8575+/−0.5, 705.3614+/−0.5, or acombination thereof. Additionally, one or more detected ions can bemonoisotopic or isotopic, non-limiting examples of which have m/z of1406.6910+/−0.5, 1407.6988+/−0.5, 1408.7067+/−0.5, 1409.7145+/−0.5,1259.6226+/−0.5, 1260.6304+/−0.5, 1261.6383+/−0.5, 1262.6461+/−0.5,1172.5906+/−0.5, 1173.5984+/−0.5, 1174.6063+/−0.5, 1175.6141+/−0.5,1075.5378+/−0.5, 1076.5456+/−0.5, 1077.5535+/−0.5, 1078.5613+/−0.5,960.5109+/−0.5, 961.5187+/−0.5, 962.5266+/−0.5, 963.5344+/−0.5,845.4839+/−0.5, 846.4917+/−0.5, 847.4996+/−0.5, 848.5074+/−0.5,758.4519+/−0.5, 759.4597+/−0.5, 760.4676+/−0.5, 761.4754+/−0.5,687.4148+/−0.5, 688.4226+/−0.5, 689.4305+/−0.5, 690.4383+/−0.5,630.3933+/−0.5, 631.4011+/−0.5, 632.4090+/−0.5, 633.4168+/−0.5,559.3562+/−0.5, 560.3640+/−0.5, 561.3719+/−0.5, 562.3797+/−0.5,472.3242+/−0.5, 473.3320+/−0.5, 474.3399+/−0.5, 475.3477+/−0.5,288.203+/−0.5, 289.2108+/−0.5, 290.2187+/−0.5, 291.2265+/−0.5, or acombination thereof.

In yet another aspect, thyroglobulin from the biological sample can bedigested to form peptide LGDQEFIK (SEQ ID 003). In this case, a detectedion can have monoisotopic or isotopic and an m/z ratio of475.2537+/−0.5, 475.7576+/−0.5, 476.2615+/−0.5, 476.7654+/−0.5, or acombination thereof. Additionally, one or more detected ions can bemonoisotopic or isotopic, non-limiting examples of which have m/z of949.4989+/−0.5, 950.5067+/−0.5, 951.5146+/−0.5, 952.5224+/−0.5,836.4149+/−0.5, 837.4227+/−0.5, 838.4306+/−0.5, 839.4384+/−0.5,779.3934+/−0.5, 780.4012+/−0.5, 781.4091+/−0.5, 782.4169+/−0.5,664.3665+/−0.5, 665.3743+/−0.5, 666.3822+/−0.5, 667.3900+/−0.5,536.3079+/−0.5, 537.3157+/−0.5, 538.3236+/−0.5, 539.3314+/−0.5,407.2653+/−0.5, 408.2731+/−0.5, 409.2810+/−0.5, 410.2888+/−0.5, or acombination thereof.

In a further aspect, thyroglobulin from the biological sample can bedigested to form peptide FPLGESFLVAK (SEQ ID 004). In this case, adetected ion can have monoisotopic or isotopic with an m/z ratio of604.3403+/−0.5, 604.8442+/−0.5, 605.3481+/−0.5, 605.8520+/−0.5, or acombination thereof. Additionally, one or more detected ions can bemonoisotopic or isotopic ions, non-limiting examples of which have m/zof 1207.6721+/−0.5, 1208.68+/−0.5, 1209.69+/−0.5, 1210.70+/−0.5,1060.6037+/−0.5, 1061.61+/−0.5, 1062.62+/−0.5, 1063.63+/−0.5,963.5510+/−0.5, 964.56+/−0.5, 965.57+/−0.5, 966.57+/−0.5,850.4669+/−0.5, 851.47+/−0.5, 852.48+/−0.5, 853.49+/−0.5,793.4454+/−0.5, 794.45+/−0.5, 795.46+/−0.5, 796.47+/−0.5,664.4028+/−0.5, 665.41+/−0.5, 666.42+/−0.5, 667.43+/−0.5,577.3708+/−0.5, 578.38+/−0.5, 579.39+/−0.5, 580.39+/−0.5,430.3024+/−0.5, 431.31+/−0.5, 432.32+/−0.5, 433.33+/−0.5, or acombination thereof.

A stable-isotope-labeled analog of the targeted peptide was synthesizedcontaining 5C₁₃/N₁₅-labeled valine (V*), PVPESKVIFDANAPV*AVRSKVPDS (SEQID 005), and six amino acids of the sequence of Tg at the amino- andcarboxy-terminuses of the tryptic digestion sites. The use of astructurally matched internal standard containing amino acids beyond thetryptic digestion sites allow improvement in assay precision bypartially compensating for variation during the sample preparation anddetection. Of particular note is that, like the target protein, theinternal standard undergoes tryptic digestion.

The doubly charged precursor ions were used for the targeted peptide andthe internal standard. Product ion mass spectrum of the VIFDANAPVAVR(SEQ ID 001) peptide is shown in FIG. 3. Multiple mass transitions ofthe targeted peptide, and the labeled internal standard, were utilizedin to facilitate high specificity of the analysis. In subject sampleswith Tg concentrations above 10 ng/mL, product ions y3, y4, y5, y6, y8provided adequate specificity, while the only mass transitions proved tobe free of interferences in samples with concentration of Tg below 10ng/mL were y3, y4, y8 (FIG. 3) and transitions corresponding to thefirst isotopes of the parent (m/z M⁺²+0.5) and the product ions y3, y4,y8 (m/z M+1). Mass transitions of the stable isotope labeled internalstandard corresponding to the above mass transitions of the unlabeledpeptide also appear to be free of interference. Specificity of theanalysis was evaluated by assessment of the ratios of the masstransitions of the targeted peptide and the internal standard. Othernon-limiting examples of potential mass transitions that could be usedin the analysis are monisotopic product ions, of non-monoisotopic parentions or non-monoisotopic product ions of non-monoisotopic parent ions.

Other mass spectrometric techniques could also be used in the analysis,including but not limited to APCI and MALDI for ionization, and the useof either MS or MS/MS using various types of mass analyzers (e.g.quadrupole, time of flight, ion trap, QTOF, Orbitrap, etc.) orcombination of thereof.

Anti-Peptide Antibody Purification

In a further aspect, the present disclosure provides a method forpurifying an antibody. Such a method can include a) providing a solutioncomprising an antibody, biological fluid, and a buffer substance; b)bringing the solution and an adsorbent (chemisorbent) with conjugatedmodified peptide (antigen) molecule in contact under conditions wherebythe antibody binds to the conjugated modified peptide attached toadsorbent; and c) recovering the antibody from the adsorbent material byusing a solution comprising a buffer substance and a salt.

In some aspects, the antibody can be captured with a modified peptide asopposed to the peptide to which the antibody was raised. Examples ofmodifications to such peptides can include, without limitation, extendedsequences or functional groups at the amino or carboxyl or both aminoand carboxyl terminuses. In another aspect, a modified peptide can be apeptide with one or more amino acids having functional groupsubstitutions with desirable properties such as hydrophobisity, 3Dstructure, etc., thus allowing antibody-antigen binding. In yet anotheraspect, the modified peptide can be a peptide with one or more aminoacids substituted with stable isotope labeled amino acids. In a furtheraspect, the modified peptide can be a peptide having extended amino acidsequences or functional groups at the amino or carboxyl, or both aminoand carboxyl terminuses, and with one or more amino acids substitutedwith stable isotope labeled amino acid or unlabeled amino acid withdesirable properties (e.g. hydrophobisity, 3D structure, etc.) allowingantibody-antigen binding. In yet a further aspect, a modified peptidecan have extended amino acid sequences at the amino or carboxyl, or bothamino and carboxyl terminuses with one or more amino acids substitutedwith other amino acids with desirable properties (e.g. hydrophobicity,3D structure, etc.) allowing antibody-antigen binding. In yet anotheraspect, a modified peptide can include extended amino acid sequences atthe amino or carboxyl, or both amino and carboxyl terminuses, whichincludes one or more amino acids substituted with stable isotope labeledamino acids.

EXAMPLES Example 1 Preparation of Reagents, Standards, and QualityControl Samples

Standard of human Tg is purchased from AbD Serotec (Oxford, UK); stocksolution of Tg is prepared at 1 mg/mL in 0.1% BSA, aliquoted in microcentrifuge tubes and stored at −70° C. Calibration standards of Tg arepurchased from Beckman Coulter (Fullerton, Calif.); Tg concentrations inthe standards are 0, 0.6, 6, 60, 150 ng/mL (0, 0.91, 9.1, 91 and 227pmol/L). Rabbit polyclonal anti-Tg antibody is purchased from Covance(Princeton, N.J.) and diluted to 60 ng/μL with 0.1% BSA. Serum qualitycontrol samples are pooled human serum samples and contain 2, 6.5, and170 ng/mL (3, 9.8, 258 pmol/L) of Tg. Working internal standard (IS) of“winged” peptide, sequence PVPESKVIFDANAPV*AVRSKVPDS (V* [13C5; 15N](SEQ ID 005); mass shift 6 Da, RS synthesis Louisville, Ky.) is preparedat a concentration of 10 pg/μL (3.95 fmol/μL) in 20% acetonitrile inwater.

Trypsin, formic acid (FA), dithiothreitol (DTT) and sodium deoxycholate(DOC) are purchased from Sigma-Aldrich (St. Louis, Mo.). All otherreagents are of highest purity commercially available. Solvents are ofHPLC grade, purchased from JT Baker (Phillipsburg, N.J.).

Example 2 Conjugation of Antibody to Magnetic Beads

Custom polyclonal rabbit anti-peptide antibody (Covance, Princeton,N.J.) is conjugated to Tosyl activated magnetic beads (DynaBeads M280,Life Technologies, Carlsbad, Calif.) according to the manufacturer'srecommendations. Briefly, the beads are washed with PBS (pH 7.4) andre-suspended in a 1M ammonium sulfate solution containing 20 μg ofantibody per milligram of beads. Beads are incubated at 37° C. for 20hours, washed and incubated for 1 hour with blocking buffer containing0.5% BSA for one hour and reconstituted to a concentration of 20 μg/μL.Antibody content on the beads is approximately 0.2 μg/μL.

Example 3 Trypsin Digestion Followed by Immunoaffinity Capture

Sample preparation is performed on a liquid handler (epMotion,Eppendorf, Hamburg, Germany). 5 μL (60 ng/μL) of rabbit anti-Tg antibodyis added to a 500 μL aliquot of serum or plasma sample and the samplesare incubated for one hour with mixing at 20° C. After the incubation,350 μL of saturated ammonium sulfate solution is added to the samples,and Tg is precipitated along with immunoglobulins (IG). The samples arevortex-mixed for 10 min, centrifuged for 5 min at 15000 g, and thesupernatants are discarded. The precipitates are reconstituted with 300μL of water, 10 μL of internal standard (IS), 10 μL of 20 mMdithiothreitol, and 30 μL of 5% sodium deoxycholate are added, afterwhich the samples are incubated at 60° C. for 30 min. After theincubation, 400 μL of 25 mM ammonium bicarbonate and 10 μL of trypsin (4μg/μL) are added to the samples, and the samples are incubated for 4hours at 37° C.

Magnetic beads are processed using a Magnetic Stand-96 (LifeTechnologies, Carlsbad, Calif.). After the digestion, 5 μL of themagnetic beads suspension is added to the samples, and the tubes areincubated with agitation at 20° C. for 8 h to allow antibody capture ofthe targeted peptide from the digest. The beads are washed with PBS (pH7.4) three times, and the targeted peptide and the digested IS areeluted with 75 μL of 25 mM glycine (pH 2). The elutions are transferredinto a 96-well plate, and 40 μL aliquots are injected on 2D LC-MS/MS.The overall time required for the sample preparation in a 96-well plateformat is approximately 20 hours.

Example 4 2D HPLC Separation

2D HPLC separation is performed on an HPLC system including series 1260and 1290 pumps (Agilent Technologies, Santa Clara, Calif.). A ZorbaxXDB-CN 50×2.1, 5 μm HPLC column is used for the 1^(st) dimensionseparation with gradient of mobile phases A 98% to 87% A in 1.3 min (A,10 mM formic acid in water; B, 10 mM formic acid in acetonitrile); the2^(nd) dimension separation is performed on a Poroshell 120 EC-C18,100×3, 2.7 μm column (both columns from Agilent Technologies) using agradient of the same mobile phases 87 to 75% A in 2 min. Thechromatographic separation is performed at 30° C.

Example 5 Mass Spectrometry Detection

Quantitative analysis is performed on an API 5500 triple-quadrupole massspectrometer with a V-spray ionization source operated in a positiveion, multiple reaction monitoring (MRM) mode. Mass transitions monitoredin the method are m/z 636.36/1059.56, 636.86/1060.56, 636.36/912.49,636.86/913.49 and 636.36/541.35 for the VIFDANAPVAVR peptide, and m/z639.34/1065.56, 639.84/1066.56, 639.34/918.48, 639.84/919.48, and639.34/547.34, for the IS. The instrument settings are adjusted tomaximize the sensitivity and the specificity of detection. The heatinggas temperature is 450° C. The settings for the nebulizing gas (air),collision gas (nitrogen), and curtain gas (nitrogen) are 40, 9, and 40.The optimized declustering potential, collision energy, collision cellexit potential, and entrance potential are 100, 30, 30, and 10V. Thedwell time for the mass transitions is 35 ms. The Q1 quadrupole is setto high resolution and Q3 quadrupole is set to unit resolution; 0.5 and0.7 Da at half height, respectively. The total analysis time per sampleis 6.5 min; the data acquisition time is started at 240 seconds, and thedata are acquired for 90 seconds. The data are processed using Analystsoftware.

Total protein concentration is measured by spectrophotometric methodusing NanoDrop 8000 (Thermo Scientific, Wilmington, Del.). Theconcentrations of IgG and albumin are measured with a commercial IA, BNII (Dade Behring, Newark, Del.) and Modular Analytics (RocheDiagnostics, Indianapolis, Ind.), respectively. The Tg-AAb test isperformed on instrument IMMULITE 2000 (Siemens, Tarrytown, N.Y.).

Example 6 Method Validation

Method validation consists of the evaluation of the imprecision,sensitivity, linearity, accuracy, recovery, carryover, ion suppression,and establishing reference intervals for Tg. Serum pools used during themethod validation are prepared from remaining aliquots of unidentifiedpatient samples submitted to ARUP laboratories (Salt Lake City, Utah)for testing. All studies with human samples are approved by theInstitutional Review Board of the University of Utah. An assessment ofwithin and between run imprecision is performed by analyzing pools ofhuman serum samples supplemented with Tg. Concentrations of Tg are 2, 6,14 and 398 ng/mL (3, 9.1, 21.2, 603 pmol/L); the samples are analyzed inthree replicates over five days; in addition three quality controlsamples (QC) samples are analyzed in routine runs over 16 days.

Sensitivity of the method is evaluated by analyzing serum samplescontaining progressively lower concentration of Tg. Five samples (range0.26-3.9 ng/mL) are prepared by serial dilution of serum pool containing3.9 ng/mL (5.9 pmol/L) with a serum pool not containing Tg, the samplesare analyzed in duplicate over two days.

Linearity of the method is evaluated by analyzing seven samples preparedby mixing in different proportions two serum pools containing 5 and 1045ng/mL (3.3 and 886 pmol/L) of Tg. The samples are analyzed in duplicateover two days. Blank samples are injected after high standards toevaluate carryover potential of the method.

Lower limit of quantification (LOQ) and upper limit of linearity (ULOL)are determined as the lowest concentrations at which accuracy is within±15%, imprecision is <15% and a ratio of the mass transitions ismaintained at ±30%. Limit of detection (LOD) is the lowest concentrationat which chromatographic peaks are present in all mass transitions andsignal to noise ratio was >5.

The method is compared with the Access Beckman Coulter DxI800 Tgimmunoassay performed at ARUP Laboratories. Two types of samples areused for the comparison: samples tested negative for Tg-AAb (n=73), andsamples tested positive for Tg-AAb (n=113). Samples with concentrationof Tg-AAb below 20 IU/mL are considered as Tg-AAb negative. Ratio of theconcentrations determined from multiple mass transitions is used forevaluation of the specificity of analysis.

Magnetic beads enrichment recovery is determined by performing affinityenrichment of a pool of digested serum samples containing 80 ng/mL ofTg. IS is added before enrichment to three samples, and to another threesamples after the enrichment. The difference between the observedconcentrations of Tg in the pre- and post-enrichment spiked samplesgives a measure of the recovery.

Sample dilution is evaluated by analyzing serum sample containing over3000 ng/mL of Tg analyzed with dilution factors (5, 10, 20, 30, and 50).

Effect of lipemia, hemolysis and icterus is evaluated by analyzing poolsof ‘normal’ serum, lipemic, hemolized and ichteric samples ‘as is’, andmixed in ratio 1:1 (normal′ serum/lipemic; ‘normal’ serum/hemolized; and‘normal’ serum/icteric) and concentrations are compared withconcentrations measured in the individual samples.

Ion suppression is evaluated using post column infusion method(Matuzevsky et al., 2003, Strategies for the assessment of matrix effectin quantitative bioanalytical methods based on HPLC-MS/MS, Anal Chem75:3019-3030, incorporated herein by reference). A set of patientsamples free of Tg is analyzed by the method, while standard ofVIFDANAPVAVR peptide (100 ng/mL, flow rate 5 μL/min) is infused into theeffluent of the analytical column. The chromatograms are inspected forsigns of ion suppression.

Blood from five volunteers is collected in potassium EDTA, sodiumheparin, serum and serum separation tubes and the samples are analyzedby the method. Concentrations observed in the samples are comparedwithin each individual.

Storage stability of thyroglobulin is evaluated with purpose of findingacceptable storage conditions. Aliquots of human serum pool are storedat room temperature, in refrigerator (4° C.), and in freezer (−20° C.);the tubes are placed into a −70° C. freezer after 1, 2, 3, 4, 8, 14, 21and 28 days of storage and analyzed in a single batch.

Reference interval study for Tg is performed with samples fromself-reported healthy adult volunteers (25 men and 25 women) and 140samples from children ages 1-17 (1, 4, 7, 10, 13, and 16 year old, 10samples from boys and 10 samples from girls of the above ages). Thevolunteers are without chronic diseases, and not on thyroid medications;the blood is collected in serum separation tubes, serum is separated,and the samples are stored at −70° C.

Example 7 Method Validation

Assay specificity is demonstrated by analyzing over 500 human serum andplasma patient samples and evaluating quality of the chromatographicpeaks and ratios of the mass transitions used. Cross-contribution of themass transitions used for Tg and the internal standard is evaluated byanalyzing plasma samples containing internal standard (100 pg/mL) insamples not containing Tg (n=5), and plasma samples containing Tg above500 ng/mL (0.76 pmol/L) in the absence of internal standard (n=10). Nosignal cross-contribution is observed on the chromatograms.

An assessment of within and between run imprecision is performed byanalyzing human serum samples containing Tg at concentrations 8, 20 and480 ng/mL. As shown in Table 1, the assay imprecision at the evaluatedconcentrations is less than 11%. The LOQ and LOD of the method are 0.5and 0.25 ng/mL, respectively; signal-to-noise ratio of the masstransition m/z 636.36/1059.56 at the LOQ is >10.

To evaluate dilution integrity, at concentrations above the upper limitof linearity of the method, subject serum samples containing 2425 ng/mLof Tg are diluted with serum not containing Tg using 5, 10, 20, 30, 50,and 100-fold dilution, and each sample is analyzed in duplicate.Concentrations observed in the samples agree with each other to within10%. Results of the method comparison with Access analyzer (BeckmanCoulter) (FIG. 5) show Deming regression IA=1.01*LC-MS/MS−1.67, r=0.974,Sy/x=9.37. The observed precision and accuracy of the method meet theacceptance criteria for diagnostic tests of small molecules. FIG. 5shows results of the LC-MS/MS method comparison with Access analyzer(Beckman Coulter) using Tg-AAb negative (A, B) and Tg-AAb positivesamples (C). (A) Concentrations 0-350 ng/mL IA=1.00*LC-MS/MS-2.3,r=0.982, Sy,x=9.5; (B) Concentrations <60 ng/mL. IA=1.01*LC-MS/MS-2.1,r=0.956, Sy,x=2.8; (C) IA=0.53*LC-MS/MS−0.1, r=0.586, Sy,x=1.1.

A set of 113 samples tested positive for Tg-AAb (Tg-AAb concentrationrange 19-1740 IU/mL; Tg recovery test results 27-97%) are analyzed byLC-MS/MS and the concentrations are compared to the concentrationsdetermined using a Beckman Coulter Access analyzer (FIG. 5C).Concentrations of Tg measured by this method are higher than valuesdetermined by immunoassay. Difference in concentrations is caused by thepresence in the samples of Tg-AAb that interferes with the immunoassay.The results suggest that the mass spectrometry based test overcomesinterference of Tg-AAb and would allow detecting recurrence of thyroidcancer in subjects having Tg-AAb earlier during the course of diseasethan the immunoassays.

Of course, it is to be understood that the above-described arrangementsare only illustrative of the application of the principles of thepresent invention. Numerous modifications and alternative arrangementsmay be devised by those skilled in the art without departing from thespirit and scope of the present invention and the appended claims areintended to cover such modifications and arrangements. Thus, while thepresent invention has been described above with particularity and detailin connection with what is presently deemed to be the most practical andpreferred embodiments of the invention, it will be apparent to those ofordinary skill in the art that numerous modifications, including, butnot limited to, variations in size, materials, shape, form, function andmanner of operation, assembly and use may be made without departing fromthe principles and concepts set forth herein.

1-20. (canceled)
 21. A method for determining a concentration of aprotein biomarker in a biological sample, comprising: providing abiological sample containing a protein biomarker, at least a portion ofwhich is bound by an endogenous autoantibody and a portion of which isunbound by the endogenous autoantibody; binding the unbound proteinbiomarker in the biological sample with an antibody specific for theprotein biomarker to form antibody-bound protein biomarker; enrichingthe antibody-bound protein biomarker and the endogenousautoantibody-bound protein biomarker to form an enriched fraction;identifying the protein biomarker in the enriched fraction; anddetermining the concentration of the protein biomarker in the biologicalsample.
 22. The method of claim 21, wherein the concentration of theprotein biomarker is derived from initially unbound protein biomarkerand autoantibody-bound protein biomarker in the biological sample. 23.The method of claim 21, wherein identifying the protein biomarker in theenriched fraction further includes: digesting the antibody-bound proteinbiomarker and the endogenous autoantibody-bound protein biomarkercomplexes; and detecting a product of digestion to identify the proteinbiomarker.
 24. The method of claim 23, wherein detecting a product ofdigestion in the biological sample further includes determining theconcentration of the protein biomarker in the enriched fraction.
 25. Themethod of claim 21, wherein enriching further includes enriching theantibody-bound protein biomarker and any endogenous autoantibody-boundprotein biomarker to form an enriched fraction.
 26. The method of claim21, wherein the antibody includes a member selected from the groupconsisting of whole antibodies, antibody fragments, and combinationsthereof.
 27. The method of claim 21, wherein enriching further includesprecipitating the antibody-bound protein biomarker and any endogenousautoantibody-bound protein biomarker in a single phase.
 28. The methodof claim 27, wherein precipitating is performed with ammonium sulfate.29. The method of claim 21, wherein enriching further includesmaintaining the antibody-bound protein biomarker and any endogenousautoantibody-bound protein biomarker in solution while biologicalmaterial unrelated to the protein biomarker is separated from thesolution.
 30. The method of claim 29, wherein enriching further includesseparation from the biological sample using a member selected from thegroup consisting of anti-biomarker antibody, protein A, protein G,protein L, anti-immunoglobulin antibody, or a combination thereof. 31.The method of claim 21, wherein enriching further includes precipitatingthe antibody-bound protein biomarker and any endogenousautoantibody-bound protein biomarker along with a chemical precipitationof immunoglobulins.
 32. The method of claim 21, wherein the proteinbiomarker is a member selected from the group consisting of insulin,cardiac troponin I, tumor suppressor protein p53, tumor-associatedantigens, anti-retinal autoantibodies, markers of age-related maculardegeneration, and combinations thereof.
 33. The method of claim 21,wherein identifying the protein biomarker includes an immune-basedmethod selected from the group consisting of RIA, ELISA, EMIT,chemiluminescence immunoassay, or a combination thereof.
 34. The methodof claim 21, wherein identifying the protein biomarker is performedusing mass spectrometry.
 35. The method of claim 34, wherein the massspectrometry is tandem mass spectrometry.
 36. The method of claim 34,wherein the mass spectrometry is high resolution and high mass accuracymass spectrometry.
 37. A method for determining a concentration of aprotein biomarker in a biological sample, comprising performing thefollowing steps in sequential order: first, providing a biologicalsample including unbound protein biomarker and endogenousautoantibody-bound protein biomarker; second, adding an immunoglobulinantibody specific for the protein biomarker to the biological samplecontaining the endogenous autoantibody-bound protein biomarker such thatthe unbound protein biomarker binds with the immunoglobulin antibody toform antibody-bound protein biomarker; third, enriching theantibody-bound protein biomarker and the endogenous autoantibody-boundprotein biomarker present in the biological sample to form an enrichedfraction of the protein biomarker, wherein the enriching step isperformed by precipitating the antibody-bound protein biomarker andautoantibody bound protein biomarker with ammonium sulfate; fourth,identifying the protein biomarker in the enriched fraction, whereinidentifying the protein biomarker in the enriched fraction furtherincludes digesting the antibody-bound protein biomarker and theendogenous autoantibody-bound protein biomarker complexes, and detectinga product of digestion to identify the protein biomarker by massspectrometry; and fifth, determining the concentration of the proteinbiomarker in the biological sample.
 38. The method of claim 37, whereinthe mass spectrometry is tandem mass spectrometry.
 39. The method ofclaim 37, wherein the mass spectrometry is high resolution and high massaccuracy mass spectrometry.